The "magic spot nucleotides" (MSNs) ppGpp and pppGpp (also: (p)ppGpp) are bacterial alarmones central to the conserved stringent response, a stress adaptation mechanism that helps bacteria adapt to stress conditions and hostile environments. Current strategies to manipulate MSN levels rely mainly on genetic or environmental approaches, which are slow and lack temporal control. Chemical tools such as photocaged MSN analogues could provide such temporal control over MSN levels. However, the high negative charge of MSNs prevents spontaneous passage through the complex bacterial cell envelope. Here, we report the synthesis of photocaged, clickable, and isotope-labeled MSN analogues and their delivery into Escherichia coli comparing different approaches. A cyclodextrin-based synthetic nucleotide transporter provides particular advantages. Upon 400nm irradiation, these probes were photo-released inside living cells, where we tracked their conversion from pppGpp to ppGpp by capillary electrophoresis mass spectrometry and studied their ability to alter growth in a (p)ppGpp mutant. This work provides the first demonstration that highly charged, photocaged MSNs can traverse the bacterial envelope, be photo-released intracellularly, and be metabolically tracked in real time. These probes lay the foundation for spatially and temporally controlled studies of MSN function and of other highly negatively charged metabolites in bacteria.

Alarmone synthesis depletes GTP levels leading to a GTP-dependent switch that controls triggered, spontaneous and antibiotic-induced persister formation in Bacillus subtilis.

Bacteria must adapt their physiology to changing environments, a process often orchestrated by nucleotide messengers such as (p)ppGpp and cyclic di-GMP (c-di-GMP). While (p)ppGpp promotes survival und...

Author summary Most bacteria adapt to stressful conditions such as nutrient limitation by synthesizing a signaling molecule, known as ppGpp, that consists of a hyper-phosphorylated GTP. Synthesis of p...

Bacteria often experience nutrient limitation and, in response, attenuate energetically costly metabolic processes like protein synthesis. At the same time, however, they stimulate the expression of a subset of proteins that facilitate survival under ...

Bacteria often experience nutrient limitation and, in response, attenuate energetically costly metabolic processes like protein synthesis. At the same time, however, they stimulate the expression of a subset of proteins that facilitate survival under ...

AbstractThe stringent response is a stress signalling pathway with links to bacterial virulence. This pathway is controlled by the nucleotide alarmone (p)ppGpp, produced in Staphylococcus aureus by three synthetase enzymes. Here, we used a panel of synthetase mutants to examine the importance of this signalling network for S. aureus survival and virulence in vivo. Using a zebrafish larval infection model, we observed that infection with a (p)ppGpp null strain attenuated virulence. Zebrafish myeloid cell depletion restored the virulence during systemic infection, indicating that (p)ppGpp is important for phagocyte-mediated immune evasion. Primary macrophages infection studies, followed by in vitro tolerance assays and RNA-seq, revealed that (p)ppGpp is required to survive stressors found within the intracellular macrophage environment, with roles for each class of synthetase, and the linked transcription factor CodY, implicated. Taken together, these results define the importance of the stringent response and each class of synthetase for S. aureus infection.

npj Biofilms and Microbiomes - (p)ppGpp imposes graded transcriptional changes to impair motility and promote antibiotic tolerance in biofilms

Pathogenic bacteria rely on the stringent response to adapt to the complex and fluctuating conditions encountered within the host. However, the mechanisms by which the stringent response senses host-i...

Protein homeostasis is perturbed at extremely high growth temperatures and without molecular chaperones or proteases. The protein synthesis-inhibiting branch of the stringent response involves elevat...